WNT16 elevation induced cell senescence of osteoblasts in ankylosing spondylitis

Abstract Background WNT16 is critical for bone homeostasis, but the effect of WNT16 in ankylosing spondylitis (AS) is still unknown. Here, we investigated whether WNT16 influences bone formation and pathophysiological changes of AS in an in vitro model. Methods The bone tissue from the facet joints was obtained from seven disease control and seven AS patients. Primary osteoprogenitor cells of the facet joints were isolated using an outgrowth method. Isolated osteoprogenitor cells from both control and AS tissues were analyzed by microarray, RT-qPCR, immunoblotting, and immunohistochemistry. The bone-forming activity of osteoprogenitor cells was assessed by various in vitro assays. β-galactosidase staining and senescence-associated secretory phenotype (SASP) using RT-qPCR were used to assess cell senescence. Results In microarray analysis, WNT16 expression was significantly elevated in AS osteoprogenitor cells compared to the control. We also validated that WNT16 expression was elevated in AS-osteoprogenitor cells and human AS-bone tissues. WNT16 treatment inhibited bone formation in AS-osteoprogenitor cells but not in the control. Intriguingly, AS-osteoprogenitor cells were stained markedly with β-galactosidase for cell senescence in WNT16 treatment. Furthermore, in an H2O2 stress-induced premature senescence condition, WNT16 treatment increased cell senescence in AS-osteoprogenitor cells and WNT16 treatment under the H2O2 stress condition showed an increase in p21 protein and SASP mRNA expression. The WNT16-induced SASP expression in AS-osteoprogenitor cells was reduced in WNT16 knockdown cultures. Conclusion WNT16 is highly expressed in AS and WNT16 treatment facilitated cell senescence in AS-osteoprogenitor cells during osteoblast differentiation accompanied by suppression of bone formation. The identified role of WNT16 in AS could influence bone loss in AS patients.

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Canonical Wnts function as potent regulators of osteogenesis by human mesenchymal stem cells

The Journal of Cell Biology ◽

10.1083/jcb.200810137 ◽

2009 ◽

Vol 185 (1) ◽

pp. 67-75 ◽

Cited By ~ 102

Author(s):

Guizhong Liu ◽

Sapna Vijayakumar ◽

Luca Grumolato ◽

Randy Arroyave ◽

HuiFang Qiao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Wnt Signaling ◽

De Novo ◽

Human Mesenchymal Stem Cells ◽

Mitogen Activated Protein Kinase ◽

Bone Homeostasis

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized β-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo.

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Production of bone formation-regulating factors by osteoclasts in vitro does not correlate with the radiographic disease progression in patients with ankylosing spondylitis

Joint Bone Spine ◽

10.1016/j.jbspin.2015.06.008 ◽

2016 ◽

Vol 83 (4) ◽

pp. 468-469

Author(s):

Gleb Slobodin ◽

Boris Slobodin ◽

Doron Rimar ◽

Regina Peri ◽

Aharon Kessel ◽

...

Keyword(s):

Ankylosing Spondylitis ◽

Bone Formation ◽

Disease Progression ◽

Radiographic Disease

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Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss

Bone Research ◽

10.1038/s41413-021-00157-x ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Martina Rauner ◽

Marta Murray ◽

Sylvia Thiele ◽

Deepika Watts ◽

Drorit Neumann ◽

...

Keyword(s):

Bone Loss ◽

Bone Formation ◽

Bone Mass ◽

Marker Genes ◽

Bone Homeostasis ◽

Bone Formation Rate ◽

Osteoprogenitor Cells ◽

Cell Parameters ◽

Female Mice ◽

Osteoblast Activity

AbstractHigh erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d−1) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

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Chronic Alcohol Reduces Bone Formation Through Inhibiting Proliferation and Promoting Aging of Endothelial Cells in Type-H Vessels

10.21203/rs.3.rs-1030987/v1 ◽

2021 ◽

Author(s):

Ao Chen ◽

Xiaoting Li ◽

Jingyu Zhao ◽

Jiawen Zhou ◽

Chunfeng Xie ◽

...

Keyword(s):

Endothelial Cells ◽

Bone Formation ◽

Bone Growth ◽

Umbilical Vein ◽

Liquid Diet ◽

Bone Homeostasis ◽

Chronic Alcohol ◽

Vessel Formation

Abstract Background: Chronic alcohol is one of the leading risk factors for male osteoporosis . Angiogenesis and osteogenesis coupled by type-H vessels coordinate the biological process of bone homeostasis to prevent osteopenia. It is unknown whether alcohol inhibits type-H-vessel-dependent bone formation. Aims: This study aimed to determine whether alcohol hampers proliferation and promotes aging of endothelial cells of type-H vessels, and whether alcohol inhibits the differentiation of bone marrow-mesenchymal stem cells (BM-MSCs) into osteoblasts through reducing the number and secretion of endothelial cells in type-H vessels. Materials and Methods: Two-month-old mice fed with alcohol liquid diet (28% of calories) or normal liquid diet for two months. The tibias were isolated and detected with X-ray and micro-CT. Paraffin-embedded or frozen tibial sections were prepared and used for immunohistochemical or immunofluorescence staining respectively in vivo . Human Umbilical Vein Endothelial Cells (HUVECs) were treated with different-concentrated alcohol for 12 hours. The conditioned medium of the above HUVECs cells was collected to culture human BM-MSCs, which were induced to differentiate into osteoblasts in vitro . Results: The alcoholic diet retarded the bone growth and lead to osteoporosis, impaired bone formation of osteoblasts, and decreased CD31 hi EMCN hi type-H-vessel formation through inhibiting proliferation and promoting aging of endothelial cells in mice. Alcohol treatment obviously increased the expression of p16, while significantly decreased the expression of Bmi-1, CDK6, Cyclin D, E2F1 and BMP2 compared to vehicle. Alcohol inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. Conclusions: Alcoholic diet impaired CD31 hi EMCN hi type-H-vessel formation through inhibiting proliferation and promoting aging of endothelial cells via Bmi-1/p16 signaling, and inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. It provides a basis for developing a new treatment strategy targeting aging endothelial cells of type-H-vessel to prevent alcoholic osteopenia.

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Progressive Development of the Osteoblast Phenotype during Differentiation of Osteoprogenitor Cells Derived from Fetal Rat Calvaria: Model for in Vitro Bone Formation

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.25.509 ◽

2002 ◽

Vol 25 (4) ◽

pp. 509-515 ◽

Cited By ~ 28

Author(s):

Noriko Yamamoto ◽

Kazuyuki Furuya ◽

Keigo Hanada

Keyword(s):

Bone Formation ◽

Osteoprogenitor Cells ◽

Progressive Development ◽

Rat Calvaria ◽

Fetal Rat

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Recombinant Irisin Prevents the Reduction of Osteoblast Differentiation Induced by Stimulated Microgravity through Increasing β-Catenin Expression

International Journal of Molecular Sciences ◽

10.3390/ijms21041259 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1259 ◽

Cited By ~ 3

Author(s):

Zhihao Chen ◽

Yan Zhang ◽

Fan Zhao ◽

Chong Yin ◽

Chaofei Yang ◽

...

Keyword(s):

Bone Formation ◽

Osteoblast Differentiation ◽

Simulated Microgravity ◽

Marker Gene ◽

Calcium Deposition ◽

Bone Homeostasis ◽

Mice Model ◽

Beneficial Effects

Background: Irisin, a novel exercise-induced myokine, was shown to mediate beneficial effects of exercise in osteoporosis. Microgravity is a major threat to bone homeostasis of astronauts during long-term spaceflight, which results in decreased bone formation. Methods: The hind-limb unloading mice model and a random position machine are respectively used to simulate microgravity in vivo and in vitro. Results: We demonstrate that not only are bone formation and osteoblast differentiation decreased, but the expression of fibronectin type III domain-containing 5 (Fdnc5; irisin precursor) is also downregulated under simulated microgravity. Moreover, a lower dose of recombinant irisin (r-irisin) (1 nM) promotes osteogenic marker gene (alkaline phosphatase (Alp), collagen type 1 alpha-1(ColIα1)) expressions, ALP activity, and calcium deposition in primary osteoblasts, with no significant effect on osteoblast proliferation. Furthermore, r-irisin could recover the decrease in osteoblast differentiation induced by simulated microgravity. We also find that r-irisin increases β-catenin expression and partly neutralizes the decrease in β-catenin expression induced by simulated microgravity. In addition, β-catenin overexpression could also in part attenuate osteoblast differentiation reduction induced by simulated microgravity. Conclusions: The present study is the first to show that r-irisin positively regulates osteoblast differentiation under simulated microgravity through increasing β-catenin expression, which may reveal a novel mechanism, and it provides a prevention strategy for bone loss and muscle atrophy induced by microgravity.

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Neutralisation of Dkk-1 protects from systemic bone loss during inflammation and reduces sclerostin expression

Annals of the Rheumatic Diseases ◽

10.1136/ard.2010.132852 ◽

2010 ◽

Vol 69 (12) ◽

pp. 2152-2159 ◽

Cited By ~ 130

Author(s):

Gisela Ruiz Heiland ◽

Karin Zwerina ◽

Wolfgang Baum ◽

Trayana Kireva ◽

Jörg H Distler ◽

...

Keyword(s):

Bone Loss ◽

Bone Formation ◽

Osteoblast Differentiation ◽

Bone Architecture ◽

Bone Homeostasis ◽

Osteocyte Death ◽

Primary Osteoblasts ◽

Systemic Bone Loss

IntroductionInflammation is a major risk factor for systemic bone loss. Proinflammatory cytokines like tumour necrosis factor (TNF) affect bone homeostasis and induce bone loss. It was hypothesised that impaired bone formation is a key component in inflammatory bone loss and that Dkk-1, a Wnt antagonist, is a strong inhibitor of osteoblast-mediated bone formation.MethodsTNF transgenic (hTNFtg) mice were treated with neutralising antibodies against TNF, Dkk-1 or a combination of both agents. Systemic bone architecture was analysed by bone histomorphometry. The expression of β-catenin, osteoprotegerin and osteocalcin was analysed. In vitro, primary osteoblasts were stimulated with TNF and analysed for their metabolic activity and expression of Dkk-1 and sclerostin. Sclerostin expression and osteocyte death upon Dkk-1 blockade were analysed in vivo.ResultsNeutralisation of Dkk-1 completely protected hTNFtg mice from inflammatory bone loss by preventing TNF-mediated impaired osteoblast function and enhanced osteoclast activity. These findings were accompanied by enhanced skeletal expression of β-catenin, osteocalcin and osteoprotegerin. In vitro, TNF rapidly increased Dkk-1 expression in primary osteoblasts and effectively blocked osteoblast differentiation. Moreover, blockade of Dkk-1 not only rescued impaired osteoblastogenesis but also neutralised TNF-mediated sclerostin expression in fully differentiated osteoblasts in vitro and in vivo.ConclusionsThese findings indicate that low bone formation and expression of Dkk-1 trigger inflammatory bone loss. Dkk-1 blocks osteoblast differentiation, induces sclerostin expression and leads to osteocyte death. Inhibition of Dkk-1 may thus be considered as a potent strategy to protect bone from inflammatory damage.

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IL-23 receptor deficiency results in lower bone mass via indirect regulation of bone formation

Scientific Reports ◽

10.1038/s41598-021-89625-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Wida Razawy ◽

Celso H. Alves ◽

Marijke Koedam ◽

Patrick S. Asmawidjaja ◽

Adriana M. C. Mus ◽

...

Keyword(s):

Bone Formation ◽

Bone Mass ◽

Cortical Bone ◽

Bone Growth ◽

Osteoclast Differentiation ◽

Bone Homeostasis ◽

Littermate Control ◽

Cortical Bone Mass

AbstractThe IL-23 receptor (IL-23R) signaling pathway has pleiotropic effects on the differentiation of osteoclasts and osteoblasts, since it can inhibit or stimulate these processes via different pathways. However, the potential role of this pathway in the regulation of bone homeostasis remains elusive. Therefore, we studied the role of IL-23R signaling in physiological bone remodeling using IL-23R deficient mice. Using µCT, we demonstrate that 7-week-old IL-23R−/− mice have similar bone mass as age matched littermate control mice. In contrast, 12-week-old IL-23R−/− mice have significantly lower trabecular and cortical bone mass, shorter femurs and more fragile bones. At the age of 26 weeks, there were no differences in trabecular bone mass and femur length, but most of cortical bone mass parameters remain significantly lower in IL-23R−/− mice. In vitro osteoclast differentiation and resorption capacity of 7- and 12-week-old IL-23R−/− mice are similar to WT. However, serum levels of the bone formation marker, PINP, are significantly lower in 12-week-old IL-23R−/− mice, but similar to WT at 7 and 26 weeks. Interestingly, Il23r gene expression was not detected in in vitro cultured osteoblasts, suggesting an indirect effect of IL-23R. In conclusion, IL-23R deficiency results in temporal and long-term changes in bone growth via regulation of bone formation.

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Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis

Pathogens ◽

10.3390/pathogens9121015 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1015

Author(s):

Salvador Vazquez Reyes ◽

Supriyo Ray ◽

Javier Aguilera ◽

Jianjun Sun

Keyword(s):

Mycobacterium Tuberculosis ◽

Lipid Membranes ◽

Effect Of Temperature ◽

In Vitro Assays ◽

Vitro Model ◽

First Time ◽

Dose Dependent Increase ◽

Dose Dependent

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.

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